Compensatory proteolytic responses to dietary proteinase inhibitors ...

Arthropod-Plant Interactions (2013) 7:259–266 DOI 10.1007/s11829-012-9240-1

ORIGINAL PAPER

Compensatory proteolytic responses to dietary proteinase inhibitors from Albizia lebbeck seeds in the Helicoverpa armigera larvae Vandana K. Hivrale • Purushottam R. Lomate Shriniwas S. Basaiyye • Neeta D. Kalve

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Received: 16 May 2012 / Accepted: 28 November 2012 / Published online: 18 December 2012 Ó Springer Science+Business Media Dordrecht 2012

Abstract Plant proteinase inhibitors (PIs) have been shown to reduce the growth rates in larvae of numerous insect species. On the other hand, insects can also regulate their proteinases against plant PIs. In the present study, we report the compensatory activities of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) gut proteinases against the PIs of Albizia lebbeck seeds. Total of ten proteinase inhibitor bands were detected in the seed extract of A. lebbeck. Bioassays were conducted by feeding H. armigera larvae on diet containing partially purified PIs from A. lebbeck seeds. Results show that larval growth and survival was significantly reduced by A. lebbeck PIs. We found that higher activity H. armigera gut proteinase (HGP) isoforms observed in the midgut of control larvae were inhibited in the midgut of larvae fed on test diet. Some HGP isoforms were induced in the larvae fed on PI containing test diet; however, these isoforms showed lower activity in the larvae fed on control diet. Aminopeptidase activities were significantly increased in the midgut of larvae fed on test diet. A population of susceptible and resistant enzymes was observed in the midgut of H. armigera, when fed on diet containing PIs

Vandana K. Hivrale, Purushottam R. Lomate and Shriniwas S. Basaiyye are equally contributed. Handling Editor: Guy Smagghe. V. K. Hivrale (&) P. R. Lomate S. S. Basaiyye N. D. Kalve Department of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University Aurangabad, Aurangabad 431004, Maharashtra, India e-mail: [email protected] P. R. Lomate Plant Molecular Biology Unit, Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, Maharashtra, India

from A. lebbeck seeds. Our initial observations indicate that H. armigera can regulate its digestive proteinase activity against non-host plant PIs, too. It is important to study the exact biochemical and molecular mechanisms underlying this phenomenon in order to develop PI-based insect control strategies. Keywords Albizia lebbeck Helicoverpa armigera Midgut proteinases Proteinase inhibitors Regulation

Introduction Lepidopteran insects are one of the most important groups of crop pests in the world. Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is a polyphagous and devastating pest of many important crop plants and responsible for heavy economic losses to agriculture. H. armigera is a highly adaptive pest and infests more than 300 plant species throughout the world (Rajapakse and Walter 2007). The key physiological characteristics that facilitate survival of H. armigera even in adverse habitats are polyphagy, high mobility, fecundity, and facultative diapause (Fitt 1989). The pest has developed resistance against many insecticides; thus, conventional approaches are unsuccessful to control the pest damage (Kranthi et al. 2002). It is important to have complete understanding of the target insect physiology and midgut biochemistry while developing the methods of insect control (Kazzazi et al. 2005). It is also important to have through knowledge on the plant compounds that can specifically restrict the growth of insect pests. Plants respond to herbivory with the production of toxins and defensive proteins that target physiological processes in the insect. Herbivore attacked plants also emit

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volatiles that attract insect predators and strengthen resistance to future threats. Plants have developed both physical and molecular strategies to limit consumption by insect pests while attracting insect pollinators. A classic example of plant–insect interactions is the production of proteinase inhibitors by plants responding to damage by lepidopteran larvae. Several studies have been performed on the interactions of H. armigera gut serine proteinases and plant proteinase inhibitors with the objective of identifying potential inhibitors of insect proteinases (Giri et al. 1998, 2003; Chougule et al. 2005; Damle et al. 2005; Srinivasan et al. 2005; Tamhane et al. 2007). A number of identified plant PIs have been shown to reduce the growth rates in larvae of many insect species (Hilder et al. 1987; Duan et al. 1996; Macedo et al. 2009). Two Kunitz-type inhibitors with activity against trypsin and papain from Pithecellobium dumosum seeds have also been isolated and characterized. Both inhibitors have been shown to be effective against digestive enzymes of larvae of diverse insect orders (Oliveira et al. 2009). Macedo et al. (2010) report the effectiveness of Adenanthera pavonina trypsin inhibitor against larvae of Anagasta kuehniella (Zeller) (Lepidoptera: Pyralidae). Parde et al. (2010) evaluated 22 different host and non-host plant protease inhibitors for in vivo inhibition of H. armigera gut pro- and proteinases, and their biological activity against the pod borer H. armigera, and indicated that non-host plant PIs are good candidates as inhibitors of the H. armigera gut pro- and proteinases. Recently, studies demonstrated that the proteinase inhibitory proteins isolated from the seeds of Acacia nilotica are effective in inhibiting the development of H. armigera and also its gut proteases (Babu et al. 2012). Although plant proteinase inhibitors have been found effective against several insects (Hilder et al. 1987; Duan et al. 1996; Damle et al. 2005), their effects are transient in most cases as insects can adapt to proteinase inhibitors by over expressing proteinase inhibitor-insensitive proteinases, or by regulating the level of existing serine proteinases, or by degrading the proteinase inhibitor (Broadway 1996; Jongsma et al. 1995; Giri et al. 1998; Patankar et al. 2001; Dunse et al. 2010). H. armigera larvae expressing high levels of a chymotrypsin can survive on a diet containing a multidomain serine PI from Nicotiana alata (Dunse et al. 2010). Our previous observations suggest that insects can regulate their digestive enzyme levels to obtain better nourishment from the diet and to avoid toxicity due to nutritional imbalance (Hivrale et al. 2011; Lomate and Hivrale 2011). The presence of a number of proteinase isoforms with diverse specificities in the midgut of phytophagous insects has great importance for the survival and adaptation on different host plants (Hivrale et al. 2005). The adaptation of pest to host plant defense compounds probably results from the selection pressure acting on an

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entire insect population when they encounter their host plants. While studying the biochemical basis of plant–insect interactions, it has been observed that the insect proteinases degrade the proteinase inhibitors (PIs) of plant, making it completely defenseless (Giri et al. 1998). Reports demonstrate the presence of several isoproteinases in the H. armigera gut possessing diverse specificity (Harsulkar et al. 1998; Lomate and Hivrale 2010). The analyses have shown the incompetence of proteinase inhibitors from various host plants against the midgut proteinases of H. armigera (Patankar et al. 2001; Chougule et al. 2003). Thus, studies have been directed toward the search of potent inhibitors from non-host plants that can specifically inhibit the midgut proteinases of insects from various orders (Harsulkar et al. 1999). Efforts have been taken and several non-host plants have been screened to identify potential inhibitors of H. armigera gut proteinases. Studies show that the proteinase inhibitors from various non-host plants are promising candidates to control the phytophagous insects. However, it is evident from the literature that reduction in serine protease activity by ingestion of non-host plant proteinase inhibitors can be compensated with a significant induction of exopeptidase activities (Lara et al. 2000; Vila et al. 2005; Lomate and Hivrale 2011). From these results it seems that H. armigera can regulate its midgut proteinase levels against nonhost plant proteinase inhibitors too. Hence, there is need to study the differential responses of insect midgut proteinases against non-host plant proteinase inhibitors in order to search and identify potent inhibitors with novel properties. In the present paper we have studied the responses of H. armigera midgut proteinases after ingestion of proteinase inhibitors from Albizia lebbeck. For this study we have selected a non-host plant A. lebbeck, since we detected several proteinase inhibitors isoforms in its seed extract. We studied the effects of H. armigera feeding on A. lebbeck plant proteinase inhibitors on: (1) larval growth, and (2) in vitro and in vivo effect of partially purified A. lebbeck proteinase inhibitors on the activities of serine proteinases and aminopeptidases. Quantitative estimations were performed using enzyme assays, while qualitative analysis was carried out using electrophoretic separation followed by in-gel visualization of the proteinase isoforms on polyacrylamide gels.

Materials and methods Materials The following chemicals used for experiments were obtained from Sigma-Aldrich (USA): leucine p-nitroanilide (LpNA), N-a benzoyl-DL-arginine p-nitroanilide (BApNA),

Responses of H. armigera midgut proteases to PIs

N-succinyl-Ala–Ala-Pro-Phe p-nitroanilide (SAAPFpNA) and gelatin. Chemicals for electrophoresis were purchased from Merck, Germany, and Sisco research laboratory (SRL), Mumbai, India. All other chemicals used were of high analytical grade. Seeds of A. lebbeck were purchased from local medicinal plant store. Extraction of seed inhibitors Seed powder was prepared by grinding decorticated A. lebbeck seeds with a mortar and pestle and defatted using hexane and Folch’s mixture (chloroform/methanol 3:1 v/v). The defatted seed powder was extracted in distilled water (1:6 w/v) containing 1 % PVP at room temperature. After 24 h, the extract was centrifuged at 9,168g for 10 min at 10 °C. The supernatant was used as crude inhibitor extract. Protein contents of the seed extract were estimated by the method of Lowry using bovine serum albumin as standard (Lowry et al. 1951).

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Feeding assays Bioassays were conducted by feeding neonate H. armigera larvae on a control diet containing defatted chickpea powder and on a test diet containing partially purified A. lebbeck PIs (1 mg/g) incorporated into defatted chickpea powder. The major ingredients of the diets were sorbic acid, ascorbic acid, methyl P-hydroxy benzoate, vitamin B-complex, and the defatted chickpea powder. Thirty neonates (for each replication) were transferred to control as well as test diet. Insects were maintained at 28 ± 2 °C, 60 % RH, and a photoperiod of 14 h light and 10 h dark. The feeding assays were continued up to 12 days, and the % mortality was recorded at 3 day intervals. Actively feeding 4th instar larvae from control and test diets were selected for further experiments. The larvae were immobilized by keeping at -20 °C for 30 min, dissected ventrally, and midguts were removed. Midgut extracts of larvae fed on each diet were pooled separately, frozen, and stored at -20 °C until further use.

Electrophoretic detection of PI isoforms Preparation of midgut extracts Albizia lebbeck PI isoforms were visualized by using 6 % native polyacrylamide gel electrophoresis (native PAGE), co-polymerized with 0.5 % gelatin. Seed extract (40 lg) was loaded on 6 % native PAGE, and electrophoresis was carried out at constant current of 20 mA. After electrophoresis, the gel was washed with distilled water and equilibrated in 0.1 M Tris–HCl buffer pH 7.8. After equilibration, the gel was placed in 0.1 % (w/v) trypsin solution (prepared in the same buffer) and incubated for 2 h at 37 °C. Then, the gel was stained with CBBR-250 and destained to visualize PI isoforms. Partial purification of PIs The extract (100 mL) of A. lebbeck seeds was subjected to acetone precipitation to concentrate the proteins. The precipitate was loaded on sephadex G-50 gel filtration column and eluted with Tris–HCl buffer pH 7.8. The PI activity in the eluted fractions was detected on X-ray film by spot test method (Hivrale et al. 2011). The positive PI activity fractions were pooled and used for the insect feeding assays. Insect culture Helicoverpa armigera larvae were obtained from International Crop Research Institute for Semi Arid Tropics (ICRISAT) Patancheru, Hyderabad, India. Larvae were maintained under laboratory conditions on an artificial diet (Srinivasan et al. 2005), and subsequent generations were used for feeding assays.

Midgut tissues of the larvae were removed, weighed, and homogenized with a pre-chilled mortar and pestle in 1:6 (w/v) volume of ice-cold 0.1 M Tris–HCl buffer pH 8.0. All the homogenates were centrifuged at 9,168g at 4 °C for 20 min. The supernatants were collected and divided into small aliquots and stored at -20 °C until use. Protein concentration in supernatants was estimated by the method of Lowry using bovine serum albumin as standard (Lowry et al. 1951). Two types of analysis were performed for zymograms and enzymatic assays. In the first analysis, serine proteinase and aminopeptidase activities were detected and assayed directly in the midgut extracts of larvae fed on control and test diets. In second analysis, the midgut extracts of larvae (40 lg) fed on control and test diets were pre-incubated (at 37 °C for 30 min) with the partially purified PIs, and zymograms and enzymatic assays were carried out. Visualization of proteinase isoforms Proteinase isoforms in the midgut extracts were electrophoretically separated on discontinuous native PAGE (4 % stacking and 8 % resolving gel) and visualized by using gelatin reverse zymography (Feliocioli et al. 1970; Lomate and Hivrale 2011). Midgut extracts (40 lg) of larvae fed on control and test diets were loaded on 8 % native PAGE, and electrophoresis was carried out at constant current of 20 mA. After electrophoresis, the gel was washed with distilled water and equilibrated in 0.1 M Glycine–NaOH buffer pH 9.6. After equilibration, the gel was placed in 1 % (w/v) gelatin (prepared in the same buffer) and incubated for

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2 h at 37 °C. Then, the gel was stained with CBBR-250 and destained to visualize proteinase activity isoforms. In-gel visualization of aminopeptidase isoforms Native slab polyacrylamide gel electrophoresis (PAGE) was used to separate aminopeptidases (Davis 1964) on discontinuous polyacrylamide gel (4 % stacking gel and 10 % resolving gel). Enzyme activity was detected by zymogram analysis (Bozic and Vujcic 2005). Midgut extracts (100 lg) of larvae fed on control and test diets were loaded on native PAGE, and electrophoresis was carried out at constant current of 20 mA. After electrophoresis, the gel was washed twice with distilled water for 10 min and equilibrated with 0.1 M Tris–HCl buffer pH 8.0, twice for 5 min. After that, the gel was dipped into 10 mM LpNA solution prepared in 0.1 M Tris–HCl buffer pH 8.0 (LpNA was initially solubilized in 800 lg of dimethyl sulphoxide and further diluted to 10 ml with buffer) and incubated at 37 °C for 20 min. Diazotization of liberated p-nitroaniline was done at 0 °C by immersing the gel into freshly made 0.1 % (w/v) sodium nitrite solution in 1 M HCl for 2 min. The excess sodium nitrite was removed with 1 % (w/v) urea, the reaction being continued for 30 s by gentle shaking of the gel. The diazotized gel then immersed into 0.025 % (w/v) 1-naphthylamine solution in 22 % (v/v) ethanol with gentle agitation until distinct pink azo dye formed (2–5 min). Trypsin, chymotrypsin, and aminopeptidase activity assays Trypsin and chymotrypsin activity assays Trypsin and chymotrypsin activity assays were carried out using selective substrates. Midgut extracts of larvae fed on control and test diets were used for the assay of trypsin and chymotrypsin activity. Respective reactions were started by adding each midgut extract (40 lg) in 200 lL of 10 mM BApNA (specific substrate for trypsin like enzymes) or SAAPFpNA (specific substrate for chymotrypsin like enzymes) prepared in 0.1 M Glycine-NaOH buffer pH 9.6 (substrates were initially solubilized in 800 lL dimethyl sulphoxide or dimethyl formamide and further diluted to 10 mL with buffer) and continued up to 30 min at 37 °C. After 30 min, the reactions were terminated by adding 150 lL of 30 % acetic acid. The rate of production of p-nitroaniline was measured at 410 nm.


200 lL of 10 mM LpNA prepared in 0.1 M Tris–HCl buffer pH 8.0 (LpNA was initially solubilized in 800 lL of dimethyl sulphoxide and further diluted to 10 mL with buffer) and continued up to 30 min at 37 °C. After 30 min, the reactions were terminated by adding 150 lL of 30 % acetic acid. The rate of production of p-nitroaniline was measured at 410 nm. Statistical analysis All the experiments were carried out at least three times with three biological and three technical replicates; Student’s t tests were performed to verify the significance of the observed differences in enzyme activities on different diets. The statistical analysis was performed using SPSS 15.0 (SPSS Inc., Chicago, IL, USA).

Results Identification of A. lebbeck proteinase inhibitors Albizia lebbeck seed extract was processed to identify proteinase inhibitor isoforms. PIs were electrophoretically separated and detected with gelatin-PAGE. Total ten PI isoforms were detected in the A. lebbeck seeds extract, and these isoforms were designated as PI 1 to PI 10 (Fig. 1). PI 3, 5, and 6 showed highest activity, whereas PI 1 and 2 observed to be lowest activity isoforms. Most of the PI activity isoforms detected in the seed extract of A. lebbeck appeared to be moderate or high activity PI isoforms (Fig. 1). The PIs from the seed extract of A. lebbeck were partially purified and used for bioassays. Feeding analysis Neonate H. armigera larvae were fed on diet containing purified PIs and change in the mortality on the control and test diets was recorded at 3-day intervals. The results of the feeding bioassays showed that no mortality was recorded for H. armigera fed on a control diet and the test diet after three days of feeding. However, significant difference was recorded between the control and test diet in terms of percentage mortality on day 6 (P \ 0.01) and days 9 and 12 (P \ 0.001) of feeding. In general, the highest mortality rate was recorded on the test diet as compared to control diet after 12 days of feeding (Fig. 2).

Aminopeptidase activity assay Detection of proteinase activity isoforms Midgut extracts of larvae fed on control and test diets were used for aminopeptidase activity assays. Respective reactions were started by adding each midgut extract (40 lg) in

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Midguts of the larvae fed on control and test diets were removed and processed to visualize the proteinase activity


Fig. 1 Detection of proteinase inhibitor isoforms from the seed extract of A. lebbeck. PIs were separated on 6 % native polyacrylamide gel containing 0.5 % gelatin. Total of ten PI activity isoforms were detected and designated as PI1 to PI10

Fig. 2 Evaluation of mortality rate of H. armigera larvae fed on diet containing partially purified PIs from A. lebbeck seeds. The feeding assays were continued up to 12 days, and the % mortality was recorded at 3 day intervals. The experiment was performed in three replicates, and each replicate contains thirty larvae. Significant differences in the larval mortality rate were calculated by Student t tests. Error bars indicate ± SD

isoforms. Total ten H. armigera gut proteinase (HGP) isoforms were detected in the midgut extract of larvae fed on control as well as test diet (Fig. 3), and these isoforms were designated as HGP 1–10. In the control larvae, the activities of HGP 2–5 were higher, whereas HGP 1, 6, and 7 appeared as lower activity isoforms. Interestingly, in the test larvae, activities of HGP 2–9 were higher as compared to control larvae. Activities of HGPs, which were lower in the control larvae, were increased in the larvae fed on test diet. However, HGPs (2–4) with high activity in the control larvae, were less present in the test larvae. We found significant induction in the activity of

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Fig. 3 Detection of proteinase activity isoforms in the midgut of H. armigera larvae fed on control and test diets. Midgut extracts (40 lg of proteins each) were resolved on 8 % native discontinuous polyacrylamide gels, and proteinase activity isoforms were detected using gelatin reverse zymography. These bands were designated as HGP1 to HGP10. The experiment was performed three times with three biological replicates

HGP 5, 6, and 9 in the test larvae as compared to control larvae. Nevertheless, the A. lebbeck PIs inhibited most of HGPs activity in the larvae fed on control as well as test diet when the midgut extracts of these larvae were preincubated with partially purified A. lebbeck PIs prior to the assay (Fig. 3). Detection of aminopeptidase activity isoforms Three prominent aminopeptidase activity isoforms were detected in the midgut extracts of larvae fed on control and test diets. These aminopeptidase activity isoforms were designated as AP1 to AP3. Among the three aminopeptidase activity, isoforms AP1 and AP3 showed more activity as compared to AP2. Activities of aminopeptidase isoforms were higher in the larvae fed on test diets as compared to the larvae fed on control diet (Fig. 4). Activities of trypsin, chymotrypsin, and aminopeptidase Significant differences in the trypsin and chymotrypsin activities were observed in the midgut extract of larvae fed on control and test diets. Trypsin and chymotrypsin activities

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Fig. 5 Determination of activities of trypsin, chymotrypsin, and aminopeptidase in the midgut of H. armigera larvae fed on control and test diets. An assay was carried out at 37 °C using selective substrates. Rate of liberation of p-nitroaniline was spectrophotometrically measured at 410 nm. The experiment was performed three times with three biological replicates. Error bars indicate ±SD

Fig. 4 In-gel visualization of aminopeptidase activity isoforms in the midgut of H. armigera larvae fed on control and test diets. Midgut extracts (100 lg of proteins each) were resolved on 10 % native discontinuous polyacrylamide gels, and aminopeptidase activities were stained with 1-naphthylamine by diazotizing the released p-nitroaniline from LpNA with sodium nitrite. Three bands of the aminopeptidase activity were observed in the each sample, and these bands were designated as AP1 to AP3

were significantly increased in the midgut extract of larvae fed on test diet as compared to control diet (P \ 0.01). However, trypsin and chymotrypsin activities were significantly inhibited in the control and test larvae when their midgut extracts were pre-incubated with partially purified A. lebbeck PIs before assay (P \ 0.01) (Fig. 5). Aminopeptidase activities were found to be increased in the midgut extracts of larvae fed on test diet as compared to control diet (Fig. 5).

Discussion Several studies on the interactions between plant proteinase inhibitors and insect digestive proteinases have demonstrated the adaptive nature of insects, and their capacity to regulate the expression of midgut digestive proteinases against various types of plant proteinase inhibitors (Jongsma et al. 1995; Broadway 1996; Giri et al. 1998; Dunse et al. 2010; Parde et al. 2010; Hivrale et al. 2011). Since insect can adapt to proteinase inhibitors from host plants, use of non-host plant proteinase inhibitors emerged as better insect control strategy (Harsulkar et al. 1998). Number of proteinase inhibitors have been characterized from the non-host plants and proven their efficacy against

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various insect pests. Proteinase inhibitors from the seeds of bitter gourd and chili have been identified as strong inhibitors of H. armigera gut proteinases, and found to retard growth and development in this species (Telang et al. 2003; Tamhane et al. 2007). However, in the present study, we report the differential expression of H. armigera midgut proteinases against the PIs of A. lebbeck. We found ten proteinase inhibitor isoforms in the extract of A. lebbeck seeds. These inhibitors were partially purified and fed to neonate H. armigera larvae. Bioassays were conducted to assess the potency of these PIs against H. armigera. Bioassay results showed that the presence of A. lebbeck in the diet significantly affected the growth and survival of the H. armigera larvae. Results suggest the potency of A. lebbeck PIs toward the inhibition of insect digestive proteinases. Similarly, several studies report the antinutritional effects of many plant PIs on various insect species (Chougule et al. 2003; Srinivasan et al. 2005; Hivrale et al. 2011). These studies indicate the effectiveness of plant PIs against insect larvae and suggest that plant PIs are potential candidates for transgenic purposes aiming to control insect herbivores. Our results are consistent with several previous reports, which demonstrated the antinutritional effect of plant protease inhibitors against variety of insect species (Giri et al. 2003; Tamhane et al. 2007; Hivrale et al. 2011). An interesting observation of the present study is the differential activities of HGPs in the larvae fed on A. lebbeck PIs. We found that some HGP isoforms were induced in the larvae fed on PI containing diet, while these isoforms showed lower activity in the larvae fed on control diet. Higher activity HGP isoforms observed in the midgut of control larvae, were less present in the test larvae. Results are consistent with a previously observed induction pattern of H. armigera protease isoforms upon feeding on soybean trypsin inhibitor (STI) (Upadhyay and Chandrashekar 2012). It is well known that several insects produce


inhibitor-insensitive proteases to counter protease inhibitors (PI) in their diet (Jongsma et al. 1995; Bown et al. 1997). Production of inhibitor-resistant proteinases as an adaptive response to PI has also been reported in H. armigera (Wu et al. 1997). Wu et al. (1997) reported the induction of elastase-like proteinase activity in the midgut of H. armigera larvae fed on transgenic tobacco plants expressing giant taro PI. H. armigera larvae fed on soybean cultivars L17 and Sahar, which contain serine protease inhibitors, showed lowest tryptic activity, but hyper-production of chymotrypsin and elastase-like enzymes (Naseri et al. 2010). Our results from this study indicate that H. armigera regulates its digestive proteinase levels against different types of PIs. This may be achieved by constitutive synthesis of a broad spectrum of proteases in the gut, or by hyper-production of existing enzymes to overcome the effect of these antinutritional factors. It would appear that there are often two populations of digestive enzymes in target pests, namely those that are susceptible to inhibition and those that are resistant to inhibitors (Bown et al. 1997; Dunse et al. 2010), and some insects respond to ingestion of plant PIs, such as soybean trypsin inhibitor (Broadway and Duffey 1986) and oryzacystatin, by hyper-producing inhibitor-resistant enzymes. H. armigera larvae are adapted to N. alata PI by a change in just two amino acids at a particular region of sensitive chymotrypsin, converting it into resistant chymotrypsin (Dunse et al. 2010). In the present study, a population of susceptible and resistant enzymes is observed in the midgut of H. armigera when fed on diet containing PIs from A. lebbeck seeds. In the present study, we also observed the differential activities of aminopeptidases of H. armigera larvae fed on diet containing A. lebbeck PIs. Aminopeptidase activities were significantly increased in the midgut of larvae fed on PI containing diet. Results are consistent with our previous findings in H. armigera (Lomate and Hivrale 2011). It is also evident from the literature that reduction in serine protease activities by ingestion of plant proteinase inhibitors can be compensated with a significant induction of aminopeptidase activities (Lara et al. 2000; Vila et al. 2005). From these studies, it seems that there exists an insect mechanism to precisely detect the diet content and adjust the levels of these important digestive enzymes. In the present study, we observed the differential regulation of H. armigera midgut proteinases against non-host plant PIs, that is, A. lebbeck PIs. Our observations from the present study indicate that H. armigera can regulate its digestive proteinase activity against non-host plant PIs too. Therefore, it is important to study the exact biochemical and molecular mechanisms underlying this phenomenon in order to develop PI-based insect control strategies. The diverse responses of H. armigera digestive enzymes to the various PIs reflect the adaptive nature of this

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polyphagous pest, and indicates that their gut serine proteinase and aminopeptidase levels are regulated based on the diet composition. A detailed biochemical and molecular analysis of the gut proteinase and aminopeptidase isoforms upon exposure of the insect to a particular PI will highlight their specific roles.

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